Development of fetal hemoglobin inducers targeting epigenetic and oxidative stress mechanisms

项目摘要 β-血红蛋白病是世界范围内最常见的遗传性血液病， 在过去的三十年中已经开发了治疗方法。诱导正常，但发育沉默 胎儿血红蛋白（HbF）表达减少贫血并改善镰状细胞病（SCD）的临床严重性。 组蛋白去乙酰化酶（HDACs）可促进胎儿γ-珠蛋白基因的沉默 红系细胞和HDAC抑制剂显示增加β-血红蛋白病患者的HbF。但 由于需要静脉内给药和它们的抗- 增殖效应该项目的重点是创造安全有效的小分子口服HbF诱导剂。 在一项研究中，我们观察了丁酸（BA）和δ-氨基乙酰丙酸的新型口服缀合物对HbF的诱导作用。 (ALA)，命名为AN-233，和一种BACH 1抑制剂，其增强了镰状红系祖细胞中NRF 2的表达 和β-YAC小鼠。接下来，关注这些小分子药物，我们将测试中心假设， 通过表观遗传组蛋白修饰和增强的NRF 2结合调节γ-珠蛋白基因转录 介导HbF诱导。具体目标1。将测试口服前药AN-233诱导HbF的预测 通过表观遗传组蛋白修饰和NRF 2激活表达，使用相关的临床前 动物模型在β-YAC和SCD转基因小鼠中进行的治疗将确定最佳口服剂量 以及诱导HbF而不产生抗增殖作用的AN 233的时间表。的表型效应 AN-233在SCD小鼠中与氧化应激相关，并将测试其抗镰状化能力。为了达到一个指标， 我们将在贫血的幼年狒狒中测试AN-233以确定HbF诱导和作用 关于造血我们将定义AN-233激活γ-珠蛋白的机制，包括表观遗传组蛋白 乙酰化和ALA在血红素合成和NRF 2活化中的作用。具体目标2。测试预测 口服BACH 1抑制剂HPP-D增强NRF 2与γ-珠蛋白启动子ARE的结合， 转录。我们将使用镰状红系祖细胞建立BACH 1抑制剂HPP-D作为HbF诱导剂 并与临床相关的药剂（包括羟基脲和地西他滨）进行联合药物治疗。 随后，研究了HPP-D通过增强NRF 2在γ-Alzheimer细胞中的结合诱导HbF的分子机制。 将评估球蛋白启动子。最后，HPP-D在临床前SCD中诱导HbF表达的能力被证实。 小鼠将建立体内功效。预期的结果包括建立特异性有效的HDAC抑制剂， 调节β-珠蛋白基因座染色质结构以有利于γ-珠蛋白转录的试剂，用于随后的临床 研究，以解决改善SCD疾病缓解治疗的未满足需求。

PROJECT SUMMARY The β-hemoglobinopathies are the most common genetic blood disorders worldwide, but limited effective treatments have been developed over the last three decades. Induction of normal, but developmentally silenced fetal hemoglobin (HbF) expression reduces anemia and ameliorates clinical severity of sickle cell disease (SCD). Histone deacetylases (HDACs) have been shown to promote silencing of the fetal γ-globin genes in adult erythroid cells, and HDAC inhibitors were shown to increase HbF in patients with β-hemoglobinopathies. But these had limitations for pharmaceutical application due to the need for intravenous administration and their anti- proliferative effects. This project focuses on creating safe and effective small-molecule oral HbF-inducing agents. In one study, we observed HbF induction with a novel oral conjugate of butyric acid (BA) and δ-aminolevulinate (ALA), named AN-233, and a BACH1 inhibitor that enhanced NRF2 expression in sickle erythroid progenitors and β-YAC mice. Next, focusing on these small-molecule agents, we will test the central hypothesis that modulation of γ-globin gene transcription through epigenetic histone modifications and enhanced NRF2 binding mediate HbF induction. Specific Aim 1. will test the prediction that the oral prodrug AN-233 induces HbF expression through epigenetic histone modifications and NRF2 activation, using relevant preclinical animal models. Treatments conducted in β-YAC and SCD transgenic mice will establish the optimal oral dose and schedule of AN233 that induces HbF without producing anti-proliferative effects. The phenotypic effects of AN-233 in SCD mice related to oxidative stress, and its anti-sickling ability will be tested. To achieve an indicator of human drug efficacy we will test AN-233 in anemic juvenile baboon to determine HbF induction and effects on hematopoiesis. We will define mechanisms of γ-globin activation by AN-233 involving epigenetic histone acetylation and the role of ALA in heme synthesis and NRF2 activation. Specific Aim 2. Test the prediction that the oral BACH1 inhibitor HPP-D enhances NRF2 binding to the γ-globin promoter ARE to activate transcription. We will establish the BACH1 inhibitor HPP-D, as an HbF inducer using sickle erythroid progenitors and conduct combination drug treatments with clinically relevant agents including hydroxyurea and decitabine. Subsequently, molecular mechanisms of HbF induction by HPP-D through enhanced NRF2 binding in the γ- globin promoter will be evaluated. Finally, the ability of HPP-D to induce HbF expression in the preclinical SCD mice will establish in vivo efficacy. Expected outcomes include establishing specific potent HDAC inhibitors and agents that modulate β-globin locus chromatin structure in favor of γ‐globin transcription, for subsequent clinical studies to address the unmet need for improved disease-modifying therapy for SCD.